Alzheimer's disease (AD) is a non-curable and fatal illness, affecting over 5 million Americans. Despite the massive and multifaceted efforts to understand the disease and find a cure, therapeutic approaches for treating AD so far have been modestly successful. Thus, there is a need for a fresh perspective on the origin and the therapy of the disease. This proposal?s premise is rooted in the discovery of the brain?s glymphatic system, i.e. a brain-wide perivascular fluid transport system analogous to the lymphatic system in peripheral tissues, which clears interstitial fluid and waste products from the brain. The glymphatic pathway is dependent on constant CSF flow through the brain parenchyma and aquaporin-4 water (AQP4) channels present in a polarized manner on the glial endfeet. Glymphatic functioning is a highly dynamic process, with modulations of its activity accompanying sleep and even change in body posture. The efficiency of glymphatic transport diminishes with age due in part to loss of AQP4 polarization. Glymphatic clearance is reduced in mice models of AD, and importantly ?-amyloid clearance is reduced prior to presence of significant AD pathology and therefore may contribute to buildup of amyloid plaques. However, so far there are no therapeutic approaches to maintain or speed up glymphatic transport as a potential therapy for prevention of AD. The current proposal, by combining the effort of two groups with complementary expertise, is aimed at understanding the role of NO/NOS on AQP4 polarization and the importance of normal cilia function for maintenance of normal parenchymal CSF transport. This carries potential therapeutic advantage as NO/NOS activity can be pharmacologically enhanced. The proposed studies are based on novel preliminary findings that 1) that lack of nNOS activity has a dramatic effect on cilia directional (CSF) flow: while in the wild type brains flow has a clear laminar pattern of movement; specifically, the nNOS mutation results in a whirling pattern, with a sharp decrease of directionality and trajectory; 2) volumetric analysis show that brain ventricles of young, adult nNOS KO animals are enlarged strongly suggesting that nNOS deficiency leads to hydrocephalus and impaired CSF flow out of the ventricular system; and 3) the levels of expression and, importantly, the polarization pattern of AQP4 in the endfeet of perivascular glial cells are affected in nNOS mutants when compared to wild type controls. Specific Aim 1 will mechanistically dissect of the roles of the NO/NOS pathway on glymphatic transport with a focus on AQP4 polarization and cilia functioning. In addition, using genetically generated mice mutants we will investigate the role of cilia function on glymphatic transport in the setting of normal NO. In Specific Aim 2 we will characterize the contribution of NO/NOS to glymphatic clearance in the aging and in an AD mouse model. Lastly, in Specific Aim 3, by modulating the NOS system with the goal of improving glymphatic clearance we will investigate the impact of the post-hoc amyloid load on brain health focusing on glymphatic transport.